The goal of this proposal is to elucidate the role of IgA and gastrointestinal lymphoid tissues (GALT) in Clostridium difficile (CD) immunity. CD is a gastrointestinal pathogen that is a major and increasing health care burden costing the United States an estimated 3 billion dollars/year and an estimated 7,752-20,000 deaths occurring annually from CD infection and disease (CDI). Risk factors for acquiring CDI include antibiotic therapy, age (>65 years), hospitalization, and failure to mount or maintain an adequate antibody response to the CD toxins. The need for safe and effective vaccines that induce neutralizing antibody to the CD toxins and reduce CDI burden is paramount. The CD toxins are produced in the colon where gastrointestinal lymphoid tissues (GALT) predominantly produce IgA, which is the major defense mechanism of the gut from pathogens and toxins. Antigen-specific IgG responses are also induced in the GALT that may contribute to serum IgG pools. No studies have identified whether IgA or GALT is required for CDI protection. The majority of candidate CD vaccines are parenteral vaccines that induce CD toxin-specific serum IgG but do not induce intestinal IgA, which may be required to obtain robust protection against CDI. Non-CD parenteral vaccines adjuvanted with the vitamin A metabolite all trans retinoic acid (RA) induce gut targeted homing of IgA antibody secreting cells and production of antigen-specific IgA. The objective of this application is to define protective immunological pathways that are induced during CDI. We hypothesize that the GALT and IgA are critical for protective immunity to CDI and that inclusion of RA with parenterally administered CD vaccines will significantly enhance protective immunity through the enhancement of mucosal IgA. This hypothesis will be addressed by answering the following questions. Aim 1: Determine whether IgA is critical to CDI protective immunity. Is IgA required for protection against CDI infection? Does RA induce colonic toxin-specific IgA and enhance CDI protective immunity? Aim 2: Determine whether colonic immune responses are important in protective immunity to CDI. Are both IgA and IgG antibody responses to CD toxins initiated in GALT? Does the absence of GALT ablate protection from CDI? The proposed studies are innovative as they challenge key facets of the prevailing model of protective immunity to CDI and at their conclusion, we will have defined for the first time the role of mucosal IgA and GALT in protective immunity to CDI and identified new approaches to enhance protection induced by parenteral vaccines through induction of intestinal IgA by adjuvants like RA. Our proposed studies promise to yield novel and important insights to CD immunity relevant to CDI therapy and targeted vaccine design.